Doc-20181223-wa0004.docx

ANALYTICAL LAB REPORT Submitted to: Dr. Muhammad Sajid Submitted by: Group No. 03 Group Members: Areeba Fatima

BSE-C-15-45

Tayyeba Razzaq

BSE-C-15-47

Rameen khan Saqib Saleem

BS-C-15-03 BSE-C-15-42

Title: A Simple Multi-Residue Method for Determination of Chloro Oxytetracyclin in Export Buffalo Meat by HPLC-UV Detector i

Table of Contents A Simple Multi-Residue Method for Determination of Oxytetracycline in Export Buffalo Meat by HPLC-UV Detector........................................................................................................ 1 1. INTRODUCTION ..................................................................................................................................... 1 1.1 ANTIBOTICS: ........................................................................................................................................ 3 1.2 ANTIBOTIC RESISTANCE: ..................................................................................................................... 4 1.3 EFFECTS IN HUMAN BEINGS: .............................................................................................................. 4 1.4 ADVOCATES FOR RESTRICING USE OF ANTIBOTICS ............................................................................ 5 1.5 MODERATE POSITIONS ....................................................................................................................... 6 1.6 SPECIFIC RESISTANCE THAT HAS BEEN INDENTIED............................................................................. 6 1.7 SIDE EFFECTS ..................................................................................................................................... 11 1.8 ANTIBOTICS USE IN ANIMALS ........................................................................................................... 12

2 .Materials and Chemicals ........................................................................................................ 15 2.1 .Procedure ......................................................................................................................................... 15 2.2 Mobile phase..................................................................................................................................... 18 2.3 Sample Extraction Optimization ....................................................................................................... 19 References: ............................................................................................................................................. 24

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A Simple Multi-Residue Method for Determination of Oxytetracycline in Export Buffalo Meat by HPLC-UV Detector 1. INTRODUCTION Many years ago buffalo meat (BabbulusBubalis) extend the word markets through export trades. Meat of this particular specie has vast acceptability due to low quantity of fat. They are increasing up to extent of nearly organic farming system without any logical use of pesticides, veterinary drugs, hormonal compounds and growth promoters, and have been given enough withdrawal period of time before slaughter. In veterinary practice tetracyclines are greatly used particularly for the treatment of atrophic space rhinitis in pigs and pneumonia. For the detection of tetracyclines microbiological methods are nonspecific except the reason that they are sensitive methods Several chemical procedures have great specificity and advantage with respect to tetracycline detection. These procedures involve high-performance liquid chromatography (HPLC) with UV detector and thin layer chromatographic (TLC). This report describes such a procedure in which liquid-liquid extraction after a good sample pretreatment step is used for the concentration of the extract and efficient clean up. This procedure is faster and simpler. However, in age of global competitive markets in post WTO era and imposition of Sanitary and Phyto-Sanitary (SPS) measures. There is an urgent need of national survey to know about residue spectrum of buffalo meat being produced. Monitoring and strict implementation of these emerging contaminants continue to demand many positive benefits like improved national nutritional value, major economical values advantages such as job creation, and improved diplomatic relationship among the member countries concerned. But this relies on our testing and other forms of inspection by either exporting or importing country or both. Antibiotics are being used in food producing animals not only to treat disease but to maintain health and promote growth. Tetracyclines are widely used antibiotics in medicines in veterinary medicine due to its vast spectrum of antimicrobial activity, availability and low price. Unauthorized use of these antibiotics, the failure to follow label directions or inappropriate withdrawal period of time before slaughtering of animals could lead to residues in food ofanimal origin, with potential adverse effects on human.

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Antibiotics are usually grouped together based on their action. Each type of antibiotic only works against certain types of bacteria or parasites. This is why different antibiotics are used to treat different types of infection. The overuse of OTC/TC/CTC in animal production or their residues in food system poses a potential allergic reaction in sensitized individuals, but a sub-therapeutic and therapeutic levels may perturb human gut microflora by introducing resistant strains and altering the metabolic activity of the microflora, its resistant microorganisms barrier effects, and its ecological balance without any identified deleterious effects. The codex appr. residue limits (MRLs) set for OTC/TC/CTC (alone or combination) are 0.1 µg/g in muscle tissues. Though considerable research has been done on the determination of TCs residues in animal tissues, no scientific data are available for buffalo meat in concern. So, its urgent to establish a monitoring program to detect TCs residues in buffalo meat. Several chromatographic methods have been employed for the monitoring of TCs in tissue samples with different detection modes such as UV- spectrophotometry, fluorescence, and mass spectrometry in the past. All these procedures used a simple clean-up step by solid phase extraction(SPE) or matrix dispersion. The use of UV detector in residue analysis has low sensitivity, while mass spectrometry still has cost fair. In general, PDA detection is sensitive and has wide scanning range. The objective of present study was to develop and validate an analytical methodology for specific and sensitive determination of TCs in buffalo meat by HPLC with liquid liquidchromatrography by following the method mentioned with modification. The compounds were extracted with Mcllvainebuffer (pH 3.85) and cleaned up with liquid liquid extraction. To enhance the precision and accuracy of the analytical method, the validation was compelled with CAC, US-FDA, Australian Pesticides and Veterinary Medicines Agencies (apvma0 and EU decision. Finally, this validated method was applied to analysis of TCs residue in buffalo meat samples.

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1.1 ANTIBOTICS: Antibiotics are a kind of antimicrobial agents used particularly for the defence against bacteria and mostly used in medical treatment of bacterial infections and certain parasitic infections. Each type of antibiotic only dowork against certain types of bacteria and parasites. 1.1.1Types: The main types of antibiotics include: 

Penicillin



Cephalosporins



Tetracyclines



Aminoglycosides



Macrolides



Quinolones etc.

Antibiotics works on two mechanisms.Some antibiotics works by killing germs. This is often done by interfering with the structure of the cell wall of the bacterium or parasite. Those are called 1.1.2 Bactericidal antibiotics. 1.1.3 Examples of Bactericidal antibiotics: 

Penicillin’s



Cephalosporins



Fluoroquinolones (Ciprofloxacin)



Glycopeptides (Vancomycin)



Monobactams



Carbapenems

Some antibiotics works by inhibiting the growth of organisms. Those are calledBacteriostatics. 1.1.4 Examples of Bacteriostatic: 

Tetracyclines



Spectinomycin 3



Sulphonamides



Macrolides



Chloramphenicol



Trimethoprim

1.2 ANTIBOTIC RESISTANCE: There has been increased concern about the use of anti-microbials in animals (including pets, livestock, and companion animals) contributing to the rise in antibiotic-resistant infections in humans. Around 70% of all antibiotics administered are used for livestock. Some drugs are used in livestock feed to prevent illnesses and or increase growth rates, but others are used as treatment for illnesses. The use of antibiotics in livestock can bring antibiotic-resistant bacteria to humans via consumption of meat and ingestion through airborne bacteria. Manure from food-producing animals can also contain antibiotic-resistant bacteria and is sometimes stored in lagoons. This waste is often sprayed as fertilizer and can thus contaminate crops and water with the bacteria. The World Health Organization has published a list, "Critically Important Antimicrobials for Human Medicine", with the intent that it be used "as a reference to help formulate and prioritize risk assessment and risk management strategies for containing antimicrobial resistance due to human and non-human antimicrobial use.

1.3 EFFECTS IN HUMAN BEINGS: The effects of antibiotic usage in livestock transferring to humans has been well documented for over 40 years. It was first documented in 1976, where a study followed a novel antibiotic being used in livestock. The bacteria in animals and workers were regularly followed to record translational effects. The findings revealed that within 2 weeks, the bacteria found in the guts of animals fed antibiotics were resistant to the new antibiotics. Additionally, the resistant bacteria had spread to farm's laborers within 6 months. The bacteria in the stool of the laborers were tested and contained more than 80% resistance to the initial antibiotic given to the livestock. Since the primary study, there have been many well-documented events showing that antibiotic usage in livestock results in direct influence of antibiotic resistance in humans. Major

bacterial

infections

in

humans

can

be

traced

back

to

livestock.

The

family Enterobacteriaceae includes many opportunistic bacteria, including E. coliwhich are 4

commonly found in livestock. Other bacteria include Klebsiella and Staphylococcus aureus. They cause infections in the urinary tract, digestive system, skin, and bloodstream, and account for a significant portion of antibiotic-resistant bacterial infections.

1.4 ADVOCATES FOR RESTRICING USE OF ANTIBOTICS The practice of using antibiotics for growth stimulation is problematic for these reasons: 

It is the largest use of antimicrobials worldwide



Subtherapeutic use of antibiotics results in bacterial resistance



Every important class of antibiotics are being used in this way, making every class less effective



The bacteria being changed harm humans

Donald Kennedy, former director of the FDA, has said "There's no question that routinely administering non-therapeutic doses of antibiotics to food animals contributes to antibiotic resistance.]David Aaron Kessler, another former director, stated, "We have more than enough scientific evidence to justify curbing the rampant use of antibiotics for livestock, yet the food and drug industries are not only fighting proposed legislation to reduce these practices, they also oppose collecting the data. In 2013, the US Centers for Disease Control and Prevention (CDC) published a white paper discussing antibiotic resistance threats in the US and calling for "improved use of antibiotics" among other measures to contain the threat to human health. The CDC asked leaders in agriculture, healthcare, and other disciplines to work together to combat the issue of increasing antibiotic resistance.Some scientists have said that "all therapeutic antimicrobial agents should be available only by prescription for human and veterinary use. The Pew Charitable Trusts have stated that "hundreds of scientific studies conducted over four decades demonstrate that feeding low doses of antibiotics to livestock breeds antibiotic-resistant superbugs that can infect people. The FDA, the U.S. Department of Agriculture and the Centers for Disease Control and Prevention all testified before Congress that there is a definitive link between the routine, non-therapeutic use of antibiotics in food animal production and the challenge of antibiotic resistance in humans.

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HSBC produced a report in October 2018 warning that the use of antibiotics in meat production could have “devastating” consequences for humans. It noted that many dairy and meat producers in Asia and the Americas had an economic incentive to continue high usage of antibiotics, particularly in crowded or unsanitary living condition.

1.5 MODERATE POSITIONS The World Organization for Animal Health has acknowledged the need to protect antibiotics but argued against a total ban on antibiotic use in animal production.

1.6 SPECIFIC RESISTANCE THAT HAS BEEN INDENTIED At this timethe most well-documented impact on humans is foodborne gastrointestinal illness. In most cases, these illnesses are mild and do not require antibiotics; though if the infectious bacteria is drug-resistant, they have increased virulence (ability to cause disease) and lead to prolonged illness. Furthermore, in approximately 10% of cases, the disease becomes severe, requiring more advanced treatments. These treatments can take the form of intravenous antibiotics, supportive care for blood infections, and hospital stays, leading to higher costs and greater morbidity, with a trend toward higher mortality. Though all people are susceptible, populations at higher risk for severe disease include children, the elderly, and those with chronic disease. Over the past 20 years, the most common drug-resistant foodborne bacteria in industrialized countries have been non-typhoidal Salmonella and Campylobacter. Research has consistently shown the main contributing factors are bacteria sourced in livestock. A 1998 outbreak of multidrug-resistant Salmonella in Denmark linked back to two Danish swine herdscoupled with the discovery of this link, there have been improved monitoring systems that have helped to quantify the impact. In the United States, it is estimated that there are approximately 400,000 cases and over 35,000 hospitalizations per year attributable to increasing resistant strains of Salmonella and Campylobacter. In terms of financial impact in the US, the treatment of nontyphoidal Salmonella infections alone is now estimated to cost $365 million per year. In light of this, in its inaugural 2013 report on antibiotic resistance threats in the United States, the CDC identified resistant non-typhoidal Salmonella and Campylobacter as "serious threats" and called for improved surveillance and intervention in food production moving forward.

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There are other bacteria as well, where research is evolving and revealing that bacterial resistance acquired through use in livestock may be contributing to disease in humans. Examples of these include Enterococcus (including E. coli 0157) and Staphylococcus aureus. For foodborne illness from E. coli, which is still not typically treated with antibiotics because of associated risk of renal failure, increasing rates of antibiotic-resistant infections have been correlated with increasing virulence of the bacteria. In the case of Enterococcus and S. aureus, resistant forms of both of these bacteria have resulted in greatly increasing morbidity and mortality in the US. At this point, there have been studies, though a limited number, that definitively link antibiotic use in food production to these resistance patterns in humans and further research will help to further characterize this relationship.

Chlorooxytetracycline Oxytetracycline

7

Clinical data 

AU: D



US: D (Evidence of risk)

Pregnancy category

of By mouth, topical (eye drop)

Routes

administrat ion ATC code

D06AA03 (WHO) G01AA07(WHO) J01AA06 (WHO) S01AA04 (WHO) QG5 1AA01 (WHO) QJ51AA06 (WHO)

Legal status Legal status  In general: ℞ (Prescription only)

Pharmacokinetic data Elimination 6–8 hours half-life Excretion

Kidney

Identifiers IUPAC name[show] CAS



79-57-2

Number PubChem C 54675779 ID

8

 DB00595 DrugBank  10482174 ChemSpide

r 

UNII

SLF0D9077S

KEGG 

D00205

ChEBI 

CHEBI:27701

ChEMBL

ChEMBL1517

 OAQ (PDBe, RCSB PDB) PDB ligand

E number

E703 (antibiotics)

ECHA Info 100.001.103 Card

Chemical and physical data Formula

C22H24N2O9

Molar mass 460.434 g/mol 3D

 Interactive image model

(JSmol)

9

SMILES InChI (verify)

10

Oxytetracycline was the second of the broad-spectrum tetracycline group of antibiotics to be discovered. Oxytetracycline works by interfering with the ability of bacteria to produce essential proteins. Without these proteins, the bacteria cannot grow, multiply and increase in numbers. Oxytetracycline therefore stops the spread of the infection and the remaining bacteria are killed by the immune system or eventually die. Oxytetracycline is a broad-spectrum antibiotic, active against a wide variety of bacteria. However, some strains of bacteria have developed resistance to this antibiotic, which has reduced its effectiveness for treating some types of infections. Oxytetracycline is still used to treat infections caused by Chlamydia (e.g., the chest infection psittacosis, the eye infection trachoma, and the genital infection urethritis) and infections caused by Mycoplasma organisms (e.g., pneumonia). Oxytetracycline is also used to treat acne, due to its activity against the bacteria on the skin that cause acne (Propionibacterium acnes). It is used to treat flare-ups of chronic bronchitis, due to its activity against the bacteria usually responsible, Haemophilus influenza. Oxytetracycline may also be used to treat other rarer infections, such as those caused by a group of micro-organisms called rickettsia (e.g., Q fever). To make sure the bacteria causing an infection are susceptible to it, a tissue sample is usually taken, for example a swab from the infected area, or a urine or blood sample. Oxytetracycline was patented in 1949 and came into commercial use in 1950.

1.7 SIDE EFFECTS Side effects are mainly gastrointestinal and photosensitive allergic reactions common to the tetracycline antibiotics group. It can also damage calcium-rich organs, such as teeth and bones, although this is very rare. It sometimes causes nasal cavities to erode; quite commonly, the BNF suggests, because of this, tetracyclines should not be used to treat pregnant or lactating women and children under 12 except in certain conditions where it has been approved by a specialist because there are no obvious substitutes. Candidiasis(thrush) is not uncommon following treatment with broad-spectrum antibiotics. 11

1.8 ANTIBOTICS USE IN ANIMALS the use of subtherapeutic levels of penicillin and the tetracyclines in animal feeds has raised the question of the effects of such practices on human health. The Food and Drug Administration (FDA) has proposed a ban on certain antibiotics at subtherapeutic levels in feed because of the potential for compromising the health of humans. A large segment of the regulated industry, including farmers and ranchers, has contended that in nearly 30 years of use, antibiotics at subtherapeutic levels in animals have not compromised human or animal health or influenced the therapy of human disease. The FDA has contracted with the Assembly of Life Sciences, National Academy of Sciences, for a review and evaluation of human health effects of antibiotics in animal feeds. The Committee to Study Human Health Effects of Subtherapeutic Antibiotic Use in Animal Feeds has been appointed to: 1. study the human health effects of subtherapeutic use of penicillin and tetracyclines (chlortetracycline and oxytetracycline) in animal feeds; 2. review and analyze published and unpublished epidemiological and other data as necessary to assess the human health consequences of the subtherapeutic use of penicillin and tetracyclines in animal feeds; and 3. assess the scientific feasibility of additional epidemiological studies, and, if needed, to make recommendations about the kind of research necessary, its estimated cost and time requirements, and possible mechanisms to be used to conduct such studies. Under the terms of the contract, subtherapeutic levels are defined as use of the agent at levels of 200 g/ton or less, and/or use of the agent for 2 weeks or longer. Animal feeds include milk replacers, medicated blocks, and liquid feeds. Critical experimental studies on the effect of lowlevel antibiotic feeding on animal therapy and human health are de finitely needed. It is proposed that studies be conducted in the following areas: 1. Does the Feeding of Tetracycline and Penicillin Compromise Animal Therapy?—This research should be done with swine, poultry, and cattle. In swine and poultry, conditions should

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be closely controlled. In cattle it would seem essential that research be conducted in commercialtype feedlots. 2. The Relationship of Antibiotic Feeding to Human Health— Although these studies are very complex and time-consuming, it is important that some effort be started in this direction. The incidence of disease-resistant organisms could be determined in humans in industries in which the workers have close contact with animals and animal products and with people who work in industries that have no contact with animals or animal products. Also the incidence of disease and the effectiveness of therapy should be studied. Some information might be obtained by surveys of existing information. 3. Mechanisms of Action of Antibiotics in Growth Promotion— Current evidence strongly suggests that the growth-promoting effect from low-level feeding of antibacterial compounds is not solely related to disease prevention. Knowledge of the mechanisms involved is a vital missing link. If known, the study of other means of eliciting a similar response would become feasible. Thus, such new knowledge would offer the potential for eliminating some or all of the current reasons for using feeding levels of antibacterial drugs. Very little use is made of penicillin and the tetracyclines for low-level feeding (5 to 10 g/ton) for stimulating growth in poultry production. A major reason for this is that these antibiotics are not approved by the FDA for use in combination with monensin (''Coban,'' an anticoccidial drug). Monessen is used in about 85 percent of the broiler feed prepared in this country. Only the antibiotics lincomycin, bacitracin, and the bambermycin’s can be used at low levels in combination with this anticoccidial drug (Anonymous 1979c). Very little penicillin is used in poultry production either for subtherapeutic or therapeutic purposes. Some penicillin is used in the diluent for Marek's disease vaccine, which is injected into day-old chicks. It is also used as a treatment for erysipelas in turkeys at a level of 100 g/ton plus a level injected to provide approximately 5 mg/kg body weight. Apparently little or no penicillin is used for treatment of diseases in chickens. The tetracycline drugs are used quite extensively for treatment of diseases and for improving suboptimal performance of birds. Frequently, tetracycline (200 g/ton) or a combination of oxytetracycline (50 to 100 g/ton) and neomycin (35 to 140 g/ton) is used in the starter feeds for both turkeys and broilers. These are used in the first 0.23 kg of feed for each bird. In the case of 13

broilers, the anticoccidial drug is removed since the combinations are not permitted by FDA regulations. The antibiotic drugs during this period of time are used to control several bacterial diseases and to get the birds off to a good start. Considerable amounts of tetracyclines are also used in laying hen diets at various times. From 50 to 100 g/ton are used for a 2-to-3-week period to improve shell quality and egg production in laying hens, particularly during the latter part of the laying cycle. Most of the feed mixed for poultry production in the United States is made by large integrated companies and in large feed mills where careful control of the inclusion of the drugs and other feed ingredients is maintained on inventories. The feed manufacturers, in using drugs in poultry rations, must follow regulations of the FDA and be inspected periodically by this agency and state feed control officials. A point that should be emphasized is that poultry producers do not indiscriminately use antibiotics in poultry feeds. Poultry operations only use antibiotics when their cost will be more than covered by the improved performance of the birds.

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2 .Materials and Chemicals  HPLC grade acetonitrile, methanol and water were obtained from, Rankem and Qualiges fine chemicals.  Water for HPLC was also obtained from Millipore water purification system was filtered through 0.22 uLfilter . disodium hydrogen phosphate (Na2HPO4) citric acid and oxalic acid were purchased from Merck and Rankem.  Oasis HLB cartbridge 6cm3 was produced from waters. pure standards of Oxytetracyline ,tetracyline and chlotetracyline as their hydrochloride were obtained from Sigma Aldrich Pvt .Ltd, individual stock standard solutions of TCs at 1 mg/ml were prepared in methanol in an amber color volumetric flask seperately and were stored at -20o C in the dark for the maximum period of 2 months. a composite working standard solutions of 5ppm, 10ppm, 15ppm, 20ppm were prepared by diluting all stock solutions with methanol.  For analysis. 0.01 M oxalic acid ,0.01 M citric acid and 0.2M disodium hydrogen phosphate buffer were prepared in Milli -Q water ant filltered through 0.22 um cellulose filter .  0.01 M methanollic oxalic acid was prepared in methanol  McIIvainbufer was prepared by mixing 278 mL of 0.1 M citric acid solution in 222 mL of 0.2 M Na2HPO4 solution and PH was adjusted to 3.85 with extra citric solution All these buffer solutions were stored at 4oC.

2.1 .Procedure  Working standard solutions of 5ppm,10ppm,15ppm,20ppm of drug was prepared.  Frozen tissue sample was thawed.  External fat was trimmed off and sample was finely diced with scissors.  The finely cut samples were blended in a high speed (5,000 rpm) tissue blender for 2 minutes.  Took a polypropylene tube and weighed 10g sample into it.  It was homogenizes with 10mL Milli-Q-water for 1.5 minutes.  Aliquote (0.5g) of homogenized sample was transferred into glass test tube.  Fortified it with 50µL of variable concentrations of the working standard solution, leaving the analytes in contact with meat sample for 30 min.

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 After 3 mL of McIIvaine buffer was added.  The mixture was vortexed at high speed.  Iincubated for 5 min at room temperature.  Centrifuged at 3,500 rpm for 10 min in a centrifuge.  2mL of Mcllavine buffer was added and extraction was repeated .  Supernatent was pooled.  Supernatent was filtered.  Liquid liqid extraction was done by using 0.01 M methanol.  Tetracyclines were eluted with 4.5 mL of 0.0 M methanolic oxalic acid (pH 1.80).  1mL of eluent was filtered through 0.22 µm nylon filter.  Vortex and centrifuge it.  Then 20 µL of the aliquot was injected into the HPLC system.

Flow Sheet

Frozen tissue sample

Thawed, diced finely

Finely cut sample

Weighed into a poly-propylene

10g portion of this sample

tube

Blended in high speed Tissue blender at 15000 rpm for 2 minutes

Add 1oml of

Homogenized for 1.5

Aliquotes (0.5mg)

Milli-Q water

minutes in a tissue

of homogenized

Homogenizer

sample in a glass tube

Add 3ml of Mcllavine

Leave for 30 minutes

Fortified with 50µL 16

buffer

for analytic to come

of variable concentr-

in contact with meat

ations of working standard solutions

Vortex the mixture at high speed

Incubate for 5 minu-

Centrifugate

tes at room temperature

at 3500rpm for 10 minutes in centrifuge

Supernatent

Supernatent was

Extraction is repeated by

was filtered

pooled

adding 2ml Mcllavine buffer

Add filtrate in 25ml methanol and liquid

Filtrate the extract

liquid extraction

oxytetracyclin was eluted with 4.5ml of 0.01M methanolic

is done

oxalic acid

1ml of elute was filtered Centrifuged

Vortex

through 0.22µm Nylon filter

20µL of aliquote was injected in HPLC system

Results and discussion I. HPLC Conditions Optimization

17

In multi-residue analysis of tetracycline compounds. In tissue samples, HPLC with UV-detector set at 350-355nm was most commonly used. But it has low sensitivity and specificity. So, HPLC with UV detector become popular, due to wide range of UV spectrum besides it has fixed maximum wavelength covered particular compound. Other researchers also performed residue analysis of tetracycline in milk by HPLC. They used diode array detector with wavelength at 365nm without any matrix interference. In present study, the maximum absorbance was set at 355 nm wavelength ranging from 340-360 nm wavelengths. At wavelength of 340 nm or below, sharpness of peak has increased significantly, but simultaneously matrices interference also increases. Similarly, The reverse effect was noted at wavelength of 360 nm. So, for residue analysis wavelength at 355 nm was fixed without any unwanted peak in its retention time. The mean retention time for OTC/ TC/CTC was 6.9, 9.2 and 12.4 min, respectively. On performing 6 parallel determinations over 3 days, the coefficient of variation (CV) of the retention time was 0.38% for OTC, 0.22%for TC and 0.48% for CTC. In this study an C8 Phenomenex column was introduced at 35°C temperature with 0.01 M oxalic acid buffer (pH 1.60)/acetonitrile/methanol as the mobile phase. The isocratic elution under the condition is employed that allows the separation of OTC, TC, and CTC provides us good resolution and no interference. Good separation and peak shape were obtained at relatively low flow rate of 0.6 mL/min.

2.2 Mobile phase For residue analyses of tetracycline. Same mobile phase was also used that contain oxalic acid buffer. In the standardization of mobile phase, initially used diluted sulfuric acid /acetonitrile by following the method of Senyuva et al.But oxytetracycline could not be separated from the C8 column. Moreover, the pH of diluted sulfuric acid was very unstable. Similarly On the other hand, mobile phase of 0.01 M oxalic acid/acetonitrile/methanol was stable. It also efficiently separate residual components. However, a small change in flow rate and the pH of mobile phase buffer in the pKa of oxytetracycline greatly affects its retention time. At high flow rate and low buffer pH, components could not be separated from the matrix interference due to reduced sensitivity and specificity of the column. So, a flow rate of 0.6 mL/min and buffer pH of 1.60 were quite suitable for the determination of TC components in the HPLC system. These are agreement with the method developed by Pena et al. while Capolongo et al. used mobile phase buffer of pH 2.3 and at flow rate of 0.5 and 1.0 mL/min, respectively.

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2.3 Sample Extraction Optimization On the facts the other acceptable McIIvaine buffer as per methodology of Kao et al. with slight modifications. The extraction with McIIvaine buffer has no unwanted effect in subsequent HPLC analysis. The effect of pH of the sample matrix on the extraction efficacy of tetracycline was evaluated using different buffer solutions with pH 2.5-5.5. A high extraction efficacy for all three tetracycline was obtained at pH values of 3.5 and 4.0, and when pH was away from these limits clearly a decrease was found. The pKa values of OTC, TC and CTC are ranging from 2.7-3.5, so if the solution pH is higher than pKa value of these compounds the molecules could be deprotonated and have a negative charge. Some researchers used polymer monolithic material to deprotonate the compounds. The ion-exchange interactions of protonated drugs increase in acidic environment is increased in the extraction efficacy. For clean up, cartridge was used that have a HLB 6 cm3 . This polymeric cartridge does not contain any sialon backbone, so for enabling them as an effective sorbent which is the problem of TCs interacting substances such as sianol is in silica-based cartridge. Moreover, HLB cartridge has strong affinity for TCs, Through hydrophobic, hydrogen bonding and cation exchange interactions. The solvents were also evaluated that used for conditioning/ washing/elution and their volume for HLB cartridges. In early

study of

standardization, it was observed for removing interfering substances the methanol followed by water

wash

was

more

efficient

than

methanol.

Fig 1.1. Chromatogram of 5ppm solution before adding the meat 19

Fig 1.2. Chromatogram of 5ppm solution after adding the meat

Fig 1.3. Chromatogram of 10ppm solution before adding the meat

20

Fig 1.4. Chromatogram of 10ppm solution after adding the meat

Fig 1.5. Chromatogram of 15ppm solution before adding the meat

21

Fig 1.6. Chromatogram of 15ppm solution after adding the meat

Fig 1.7. Chromatogram of 20ppm solution before adding the meat

22

Fig 1.8. Chromatogram of 20ppm solution after adding the meat

23

References: 1. www.sciencedirect.com 2. www.googlescholar.com 3. www.ACSpublications.com 4. AOAC (2000). Official methods of analysis, 17th ed. Maryland, USA: Association of Official Analytical Chemists (995.09). 5. Blanchflower, W. J., McCracken, R. J., Haggan, A. S., & Kennedy, D. G. (1997). Confirmatory

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